Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis

Abstract

Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2Rexpressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100−200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.

Advances in engineered nanosystems: immunomodulatory interactions for therapeutic applications

“…In this regard, the IL-2 cytokine can be used as a surface-modifying ligand to mediate IL-2R-dependent T-cell targeting and delivery of immunotherapeutic agents. 41 Chemical species such as hyaluronic acid, folic acid, and biotins have been engineered onto the NP surface to specifically bind with the respective CD44 and biotin receptors that are overexpressed in cancer cells. Thus, modifying the particle surfaces with these ligands can selectively eliminate the cancer cells while minimally affecting the normal and healthy cell population.…”